增强生物安全与聚合物复合物Ecotropic慢病毒的人体细胞的转导

Summary

慢病毒是一个有价值的研究工具，为探索基因功能，然而，研究人员可能希望避免生产pantropic慢病毒，已知的编码或怀疑致癌基因。作为替代方案，我们目前使用ecotropic慢病毒修改的人体细胞表达ecotropic的受体mSlc7a1一个更安全的协议。

Abstract

干细胞和肿瘤细胞生物学的研究往往需要与已知或怀疑有致癌基因的人类细胞中的病毒转导，提高实验室工作人员的主要的安全问题。 Pantropic慢病毒，如常用VSV – G伪的，是一个有价值的工具，为研究基因的功能，因为他们可以转导多种细胞类型，包括非分裂细胞。然而，研究人员可能希望避免生产和离心pantropic病毒编码的癌基因，由于较高的生物安全水平的处理要求和安全问题。几个强有力的癌基因，包括c – Myc和SV40大T抗原，被称为诱导多能干细胞（IPSC），以提高生产。所有其他已知的IPSC -诱导的基因变化（OCT​​4，SOX2，KLF4，NANOG，LIN28，和p53功能的丧失）也有癌症的链接，以及相对高的安全问题。

虽然这些与癌症有关的的病毒是有用的细胞重新编程和多能性研究，他们必须使用安全。为了解决这些生物安全问题，我们证明ecotropic慢病毒的人体细胞传导的方法，进一步强调降低成本和方便处理。我们产生足够高滴度ecotropic的慢病毒转导大于90％受体表达人类细胞暴露在病毒，验证了这种方法的疗效。

慢病毒往往集中超速离心法，但是，这个过程需要几个小时的，并且能够产生气溶胶传染给人类生物医学研究人员。作为替代方案，病毒颗粒可更安全地沉淀到细胞与硫酸软骨素及聚凝胺（CS / PB）的络合。这种技术增加了功能的病毒滴度，细胞稳定表达小鼠逆转录病毒的受体，可以忽略不计时间和成本的3倍。使用CS /铅浓度比以前报道癌细胞株的最优值低约4倍，这表明，聚合物浓度应滴定利益的靶细胞类型，是最大限度地提高了人类的皮肤成纤维细胞（HDFS）传导。因此，我们描述使用methylthiazolyldiphenyl四唑溴化物（MTT）聚合物中的一个新类型的细胞毒性检测。我们观察后，无论是使用聚合物络合或聚凝胺（PB，6微克​​/毫升）的标准剂量的病毒传导HDFS相当于可行性，表示最小的急性毒性。

在这个协议中，我们描述了使用ecotropic慢病毒在人体细胞癌基因的过度表达，减少生物安全风险和提高转导率。我们也证明了高分子络合使用，以提高转导，同时避免形成气溶胶病毒颗粒的离心。

Protocol

1. Lentivirus production, harvest, and freezing Consult your institutional safety official before beginning this protocol, and follow their recommended safety guidelines. Day 1. Start with healthy, rapidly growing 293T cells to produce virus. Plate the cells at 5 x 106 cells per 10 cm plate. Use antibiotic-free 293T medium (high-glucose DMEM with 10% FBS and 4 mM L-glutamine) for virus production. Day 2. In the late afternoon, transfect 293T cells as follows (numbers given are for one 10 cm plate). Let all reagents warm up to room temperature. Pipette 375 μl OptiMEM into a microcentrifuge tube, then add 25 μl Fugene HD. Do not allow undiluted Fugene to contact the surface of the tube. In a microcentrifuge tube, mix: 5 μg transfer plasmid (Slc7a1, target vector, or fluorescent control vector) 3.75 μg packaging plasmid (pCMV-dR8.91 or psPax2) 1.25 μg envelope plasmid (pMD2.G for pantropic, pHCMV-EcoEnv for ecotropic) serum-free OptiMEM to 100 μl Combine the two tubes and incubate the mixture 20-30 minutes at room temperature. Change the medium on the 293T cells to 10 ml fresh antibiotic-free 293T medium. Add the Fugene/plasmid mix drop-wise to the plate and incubate overnight at 37°C, 5% CO2. Day 3. Change medium on the 293T cells to 10 ml fresh antibiotic-free 293T medium. Be gentle, because 293T cells adhere only loosely and can slough off by media pipetted too forcefully onto the monolayer. Incubate for two days in a humidified incubator for virus production. Day 5. Harvest virus and filter with a 0.45 mm low protein binding filter. Use immediately or dispense into single-use aliquots and freeze at -80°C. Frozen pantropic and ecotropic virus should be re-titered after it has been stored for six months and one month, respectively. Titer the virus as follows, using cells that are relatively amenable to transduction. Transduce cells with fluorescent control virus overnight using serial dilutions (e.g. 1:10, 1:100, 1:1000) in fresh medium with 6 μg/ml PB. Change to fresh medium the next day. Allow the cells two days after transduction to begin expressing the fluorescent protein, then determine the fraction of transduced cells by FACS. Calculate the titer in transforming units (TU) per ml, based on dilutions that yield < 15% transduction to minimize multiple transduction events. 2. Transduction of human target cells with murine retrovirus receptor Slc7a1 Select a multiplicity of infection (MOI) = 2 to ensure that most cells will be transduced. For target cells that are resistant to transduction such as HDFs, a higher MOI will be required; we dilute the viral supernatant only 1:2 to achieve transduction of a majority of the cells. Transduce target cells overnight with viral supernatant diluted in fresh culture medium with 6 μg/ml PB, which increases transduction by enhancing electrostatic interaction between the virus and target cell.1 Ideally one should use a minimal volume of virus, such as 1 ml for a 35 mm plate, because diffusion is a limiting factor in transduction efficiency. Culture cells for at least 48 hours after removing virus before transducing them with ecotropic virus, in order to ensure sufficient expression of the Slc7a1 receptor. (Optional) It is possible to use blasticidin to select for stably transduced cells expressing Slc7a1, if desired. A blasticidin kill curve should be generated in advance to determine the lowest effective concentration, generally between 2 – 10 μg/ml, that kills all untransduced target cells in 7 days. About 2 days after removing the Slc7a1 virus, switch the transduced cells to medium containing blasticidin. Culture the cells in blasticidin-containing medium for 7 days, at which point all remaining cells will stably express the retrovirus receptor at which point they can be transduced with ecotropic virus as well as banked for future use as a stable Slc7a1-expressing line. 3. Polymer complex titration to determine toxicity Day 1. Plate cells at 5000 cells/well in a 96-well plate, allowing at least triplicate wells for each experimental group. Include untransduced cells in complete medium to provide a baseline value for healthy cells, as well as samples consisting of cells in serum-free medium to induce growth arrest as a control. In parallel, plate cells in an appropriate format (such as a 24-well plate) for microscopic or FACS-based analysis of transduction efficiency in each experimental condition. Day 2. Transduce cells in plates set up for both the MTT and FACS analyses with virus encoding GFP, at an MOI of 0.5 to transduce ~40% of target cells. Test varied concentrations of CS/PB (e.g. 50, 100, 200, 400, 600, and 800 mg/ml of each component), as well as 6 μg/ml PB, to determine optimal amounts. Generate polymer complexes as described in Part 4, below. Day 3. Remove virus-containing medium and replace with fresh medium. Return the plates to a humidified incubator. Day 5. Analyze FACS plate to determine the transduction efficiency with each polymer concentration. Day 6. Remove the medium from all wells of the MTT plate and replace with 100 μl MTT solution (1 mg/ml MTT in complete medium). Culture for three hours in a humidified incubator to allow MTT to be reduced in the mitochondria of metabolically active cells. Remove MTT solution and replace with 200 ml MTT solvent (0.1 N HCl, 0.1% Igepal CA-630 in isopropanol). Incubate for two hours at room temperature or until all of the purple MTT formazan precipitate is dissolved. Read the absorbance on a microplate reader at 570 nm, subtracting the background reading at 690 nm. Select the optimal polymer concentration that produces maximum enhancement of transduction, as determined by FACS, with minimal effect on metabolic activity, as determined by MTT. For HDFs, we selected 100 μg/ml CS/PB based on the data shown in the Representative Results, below. 4. Ecotropic transduction with polymer complexation Prepare sterile-filtered stock solutions of PB and chondroitin sulfate at 20 mg/ml in water. Aliquot and store at -20°C. Pipette viral supernatant into a microcentrifuge tube and dilute to the desired final concentration (e.g. MOI of 2) with fresh culture medium. Add equal volumes of PB and chondroitin sulfate (concentrations determined in step 3 above) in succession, flicking the tube to mix after each addition. The viral mixture will immediately become cloudy as precipitates form. Incubate the viral mixture at room temperature for 5 minutes to allow complex formation. Remove media from the receptor-expressing target cells, replace with the viral mix, and incubate overnight at 37°C, 5% CO2. After removing the viral mix, wash the surface of the cells twice with PBS to aid in removing viral complexes. Complete removal is not necessary; we have observed no adverse effects on cell health from residual polymer complexes. 5. Verifying specificity of ecotropic transduction When producing ecotropic virus for the first time, it is prudent from a safety perspective to verify that the virus is unable to transduce unmodified human cells. Transduce human cells with ecotropic lentivirus at relatively high concentration (only a 1 to 2-fold dilution of viral supernatant in fresh medium) overnight with 6 μg/ml PB or optimized CS/PB concentration. Remove virus-containing medium and replace with fresh medium. After incubating cells for 2 days, validate transgene expression by FACS using fluorescent vectors and/or by RT-PCR with transgene-specific primers. 6. Representative results: Fig. 1A shows the lentivirus production process, and Fig. 1B shows transduction of human cells with ecotropic lentivirus, including pre-transduction with murine retrovirus receptor Slc7a1. For titering virus, we use a human rhabdomyosarcoma cell line stably transduced with Slc7a1 (Slc-hRMS). When using fluorescent control vectors to monitor transduction efficiency, we routinely achieve > 90% transduction efficiency of Slc-hRMS with ecotropic virus, as shown in Fig. 2A and 2B. Transduction rates of HDFs are generally lower because the cells have not been blasticidin-selected for receptor expression (Fig. 2C). Titers of ecotropic lentivirus are generally 10-20% of VSV-pseudotyped virus when measured on Slc-hRMS (Fig. 2D). One freeze-thaw cycle reduces titer of ecotropic virus by 16 – 3%, equal to titer loss during a single freeze-thaw cycle of VSV-pseudotyped virus (p > 0.05). Contrary to previous reports,2 we observe no positive effect on virus titer from flash freezing in dry ice. Rather, we achieve higher post-thaw titers of either pseudotype by simply placing tubes of virus into a -80°C freezer (data not shown). The MTT viability assay allows sensitive detection of growth arrest in target cells. Transduction with virus plus concentrations of CS/PB up to 800 μg/ml has no effect on HDF metabolism (Fig. 3A). Exposure to CS/PB without virus also has no toxic effect on cells (data not shown). FACS analysis of HDFs transduced with virus plus various concentrations of CS/PB shows enhancement of transduction compared to PB alone (Fig. 3B). The maximum enhancement occurs at 100 μg/ml CS/PB (Fig. 3C), several-fold lower than previously reported values.3 Thus, it is important to optimize conditions for any given target cell type. Complexation with CS/PB enhances the observed titer roughly 3-fold in Slc-hRMS (Fig. 4A, p < 0.01). In practice, this yields a greater effect on transduction efficiency at low virus concentrations than at higher concentrations (Fig. 4B), which is most likely due to multiply transduced cells and receptor saturation at higher virus concentrations. Transduction with ecotropic virus is specific for cells expressing murine retrovirus receptor. When transducing unmodified human cells with ecotropic virus, we have not observed fluorescence greater than the untransduced background whether using PB or CS/PB, as shown in Fig. 5A. Microscopically, HDFs show no transduction in the absence of receptor (Fig. 5B) while pre-transduction with receptor results in fluorescent cells (Fig. 5C). Figure 1. Schematic view of (A) the virus production process and (B) transduction of human cells with murine retrovirus receptor followed by ecotropic lentivirus. Figure 2. High-efficiency transduction of human cells with ecotropic lentivirus encoding GFP. Cells were pre-transduced with murine retrovirus receptor Slc7a1. (A) Human rhabdomyosarcoma cell line blasticidin-selected for Slc7a1 (Slc-hRMS), transduced (blue) or untransduced (pink) with GFP. (B) Fluorescent micrograph of ecotropic-transduced Slc-hRMS (4X). (C) HDFs transduced (blue) and untransduced (pink) with ecotropic GFP lentivirus; cells were transduced with Slc7a1 two days prior to ecotropic transduction. (D) Titer of VSV-G pseudotyped and ecotropic lentivirus, measured on Slc-hRMS. Figure 3. Optimizing chondroitin sulfate/polybrene (CS/PB) concentrations in human dermal fibroblasts. (A) Viability of HDFs measured by MTT assay after transduction in the presence of PB only or with varied concentrations of CS/PB. (B) Enhancement of transduction efficiency in HDFs by different concentrations of CS/PB, measured by FACS. (C) Quantification of transduction enhancement, showing that maximum transduction occurs at 100 μg/ml CS/PB. Figure 4. Effect of 400 μg/ml CS/PB on transduction of Slc-hRMS with ecotropic lentivirus. (A) Titer enhancement, and (B) fold change in transduction rate compared to 6 μg/ml polybrene alone. Figure 5. Lack of ecotropic transduction of HDFs in the absence of murine retrovirus receptor Slc7a1. (A) FACS plot of BJ human fibroblasts, untransduced (pink) or transduced (blue) with ecotropic GFP lentivirus in both cases in the absence of receptor Slc7a1. BJ human fibroblasts transduced with ecotropic GFP lentivirus, without (B) and with (C) pre-transduction with receptor Slc7a1 (4X).

Discussion

莫洛尼鼠白血病病毒（MLV）和其受体mSlc7a1的基础上重组ecotropic gammaretrovirus很好的研究和广泛使用，已使用超过20年提供的转基因小鼠细胞。最近Ecotropic gammaretrovirus也被用来提供对人体细胞的原癌基因在细胞重新编程的情况下，mSlc7a1使用amphotropic窝藏人类癌基因的病毒，以避免^产生是公认的4,5然而，慢病毒提供了显着的优势超过gammaretrovirus在转导难治性细胞群^，包括原代细胞，往往是理想的目标进行重新编程，因为慢病毒的预^集成的复杂允许7转导非分裂细胞。

已经产生不同的伪几十个，包括MLV在努力改变嗜性病毒，毒性和其他属性。已⁸ MLV的假型ecotropic慢病毒转导小鼠细胞中^，9，但已很少被用于对人体细胞的慢病毒， ¹⁰因此，我们建议使用假型MLV – ecotropic慢病毒作为一种安全，成本效益，高效的车辆提供已知或怀疑有致癌基因，包括细胞重新编程因素 ​​，对人体细胞。

要注意，这个协议并没有完全消除需要制作和使用pantropic慢病毒，相反，这个协议从pantropic病毒分离的癌基因（S），绝缘与癌症相关的病毒研究人员从潜在的自我接种，这是关键。广泛表达的蛋白质mSlc7a1和人类的同源hSlc7a1氨基酸转运与任何已知的致瘤性或赋予受体细胞的选择性生长^优势的能力，11 mSlc7a1纳入amphotropic病毒的风险相对较低。这增加的步骤可能会特别有用，在缺乏专门的病毒或组织文化设施所需要的生物安全水平的实验室。

在某些情况下，它可能是可能完全消除pantropic病毒转染质粒Slc7a1直接进入靶细胞的使用，然而，许多细胞，将这种技术非常有用的目标也难治性转染。作为一种替代方法，隔离Slc7a1能力转导的细胞杀稻瘟选择一次，VSV – G假型慢病毒可用于生成一个受体表达的细胞，ecotropic病毒后可用于常规转导这些细胞对于许多股票实验。研究人员应始终遵循与任何慢病毒工作的机构的安全指引，无论其向性。

这个协议取得ecotropic病毒滴度比VSV的假型病毒，一般为10-20％的pantropic病毒滴度与以往的研究一致，适度^降低这些滴度分别在人体细胞中测稳定与Slc7a1转，所以较低的观察ecotropic病毒滴度，部分原因可能是由于不同的靶细胞受体基因的表达。尽管如此，对于大多数应用已经足够，在我们的协议中所取得的病毒滴度，并导致在某些情况下，多数细胞的转导，细胞> 90％。

基于离心技术常用集中的病毒颗粒。然而，离心需要几个小时，产生传染性气溶胶，并可能导致重大损失的病毒颗粒，作为替代方案，络合与CS / ^PB 12可用于没有改变病毒嗜增强^转 3，此方法是快速（5分钟）和廉价（每10毫升病毒0.03元），而大约三倍观察滴度。需不需要特殊的设备或专有试剂，并在与其他细胞系的前期工作的协议，我们观察后HDFS接触到CS / PB最小^的急性毒性，13本议定书的一个潜在的缺点是，一些微观可见的高分子复合物坚持文化中的数天的细胞。我们不能排除这种可能性，这些复合物，而没有明显细胞毒性，可能对细胞过程的更微妙的影响。

在这里，我们所描述的安全和有效地转导与致癌因素的人体细胞的方法。这种做法应包括iPS细胞癌基因和干细胞生物学研究的伟大事业。

Materials

Name of the reagent	Company	Catalogue number	Comments
pLenti6/UbC/mSlc7a1	Addgene	17224	Murine MLV receptor
pMD2.G	Addgene	12259	VSV-G envelope
pCMV-dR8.91	D. Trono lab14		Packaging plasmid (equivalent plasmid psPax2 is available from Addgene, Cat. Number 12260)
pHCMV-EcoEnv	Addgene	15802	Ecotropic envelope
FUGW	Addgene	14883	GFP control plasmid
OptiMEM	Invitrogen	31985
Fugene HD	Roche	04 709 705 001
Hexadimethrine bromide (Polybrene)	Sigma-Aldrich	H9268
Chondroitin sulfate sodium salt from shark cartilage	Sigma-Aldrich	C4384
Blasticidin S	Fisher	BP 2647100
Methylthiazolyldiphenyl-tetrazolium bromide (MTT)	Sigma-Aldrich	M2128
Igepal CA-630	Sigma-Aldrich	I3021